Peripheral mechanisms of tolerance and immunoregulation are essential to prevent the damage potentially caused by autoreactive T cells that escape thymic tolerance induction. Among these, Treg cells expressing the FoxP3 transcription factor are the best characterized, with their ability to suppress immune and autoimmune responses, and their exciting potential for human therapy. They represent a constant proportion of CD4+ cells in lymphoid organs of a given individual, but vary markedly between individuals in a species. Beyond known roles for IL2 and TGF?, the mechanisms and genetic variation that control the selection and homeostatic balance of the Treg pool remain unclear. Following studies on the survival and homeostasis of conventional T cells in the previous cycle, we propose to build on our recent work that dissected the Treg transcriptional signature, established that the thymic differentiation and peripheral homeostasis of Treg cells are under independent genetic control, and showed that the extra-lymphoid localization of Treg cells are conditioned by TCR sub-repertoires and sub-phenotypes. We propose to study: 1) Cellular mechanisms of homeostatic control of Treg cells and their partners. We will continue to assess Treg population dynamics, also extending our results on extra-lymphoid populations, and in particular Treg cells which infiltrate the injured muscle and aid controlling its regeneration. We will also analyze the mechanisms of the interplay between Treg and NK cells in autoimmune lesions 2) Secondary conversion to Treg phenotype: significance and specificity. Neo-conversion of mature CD4+ lymphocytes to a FoxP3+ state can be achieved by chronic antigen stimulation, lymphopenia-driven homeostatic expansion in vivo, or IL2+GF? in vitro. Secondary Treg differentiation may be important for tolerance to self or to gut flora, and is now well established. We are attempting to work out the specificity of these cells (in polyclonal settings, non- transgenic), in terms of TCR repertoire and of transcriptional signature, and as a function of variation in the gut microbiome. 3) Molecular determinism of Treg cell homeostasis. We will build upon our description of the Treg genetic switch, in which the Treg transcriptional signature can be stably switched on, in a self- reinforcing mode, by expression of FoxP3 and any of several co-factors. We will analyze in more detail the requirements for this switch. We will explore the interactions of FoxP3 with other TFs in this context, in particular in an RNAi screen which will probe the requirement for specific cofactors in this switch behavior. RELEVANCE (See instructions): These experiments will explore the cellular mechanisms and regulatory pathways that lead to variations in the differentiation and homeostasis of FoxP3+ T regulatory cells. The results should lead to a better understanding of this population that is key to immune regulation, and impacts several autoimmune and inflammatory diseases.